PTPmu suppresses glioma cell migration and dispersal.

The cell-surface receptor protein tyrosine phosphatase mu (PTPmu) is a homophilic cell adhesion molecule expressed in CNS neurons and glia. Glioblastomas (GBMs) are the highest grade of primary brain tumors with astrocytic similarity and are characterized by marked dispersal of tumor cells. PTPmu expression was examined in human GBM, low-grade astrocytoma, and normal brain tissue. These studies revealed a striking loss of PTPmu protein expression in highly dispersive GBMs compared to less dispersive low-grade astrocytomas and normal brain. We hypothesized that PTPmu contributes to contact inhibition of glial cell migration by transducing signals in response to cell adhesion. Therefore, loss of PTPmu may contribute to the extensive dispersal of GBMs. The migration of brain tumor cells was assessed in vitro using a scratch wound assay. Parental U-87 MG cells express PTPmu and exhibited limited migration. However, short-hairpin RNA (shRNA)-mediated knockdown of PTPmu induced a morphological change and increased migration. Next, a brain slice assay replicating the three-dimensional environment of the brain was used. To assess migration, labeled U-87 MG glioma cells were injected into adult rat brain slices, and their movement was followed over time. Parental U-87 MG cells demonstrated limited dispersal in this assay. However, PTPmu shRNA induced migration and dispersal of U-87 MG cells in the brain slice. Finally, in a mouse xenograft model of intracranially injected U-87 MG cells, PTPmu shRNA induced morphological heterogeneity in these xenografts. Together, these data suggest that loss of PTPmu in human GBMs contributes to tumor cell migration and dispersal, implicating loss of PTPmu in glioma progression.

Glyoxalase I activity and immunoreactivity in the aging human lens.

Glyoxalase I (GLOI) is the first enzyme of the glyoxalase system that catalyzes the metabolism of reactive dicarbonyls, such as methylglyoxal (MGO). During aging and cataract development, human lens proteins are chemically modified by MGO, which is likely due to inadequate metabolism of MGO by the glyoxalase system. In this study, we have determined the effect of aging on GLOI activity and the immunoreactivity and morphological distribution of GLOI in the human lens. A monoclonal antibody was developed against human GLOI. GLOI immunoreactivity was strongest in the anterior epithelial cells and weaker in rest of the lens. Cultured human lens epithelial cells showed immunostaining throughout the cytoplasm. In the human lens, GLOI activity and immunoreactivity both decreased with age. We believe that this would lead to promotion of MGO-modification in aging lens proteins.

BCCIP associates with the receptor protein tyrosine phosphatase PTPmu.

The receptor protein tyrosine phosphatase PTPmu belongs to a family of adhesion molecules that contain cell-cell adhesion motifs in their extracellular segments and catalytic domains within their intracellular segments. The ability of PTPmu both to mediate adhesion and exhibit enzymatic activity makes PTPmu an excellent candidate to transduce signals in response to cell-cell adhesion. In an effort to identify downstream signaling partners of PTPmu, we performed a modified yeast two-hybrid screen using the first tyrosine phosphatase domain of PTPmu as bait. We isolated an interacting clone encoding BRCA2 and CDKN1A interacting protein (BCCIP) from a HeLa cell library. BCCIP is a p21 and BRCA2 interacting protein that has been shown to play roles in both cell cycle arrest and DNA repair. In this manuscript, we confirm the interaction between BCCIP and PTPmu identified in yeast using in vitro biochemical studies and characterize BCCIP as a PTPmu binding protein. We demonstrate that BCCIP is phosphorylated by the Src tyrosine kinase and dephosphorylated by the PTPmu tyrosine phosphatase in vitro. Furthermore, we show that BCCIP is required for both the permissive and repulsive functions of PTPmu in neurite outgrowth assays, suggesting BCCIP and PTPmu are in a common signal transduction pathway.

Rho GTPases regulate PTPmu-mediated nasal neurite outgrowth and temporal repulsion of retinal ganglion cell neurons.

Members of the receptor protein tyrosine phosphatase (RPTP) subfamily of cell adhesion molecules (CAMs) mediate neurite outgrowth and growth cone repulsion. PTPmu is a growth permissive substrate for nasal retinal ganglion cell (RGC) neurites and a growth inhibitory substrate for temporal RGCs. In this manuscript, we demonstrate that the distinct PTPmu-dependent phenotypes of nasal outgrowth and temporal repulsion are regulated by Rho GTPases. The role of Rho GTPases in the regulation of nasal outgrowth and temporal repulsion was tested by utilizing dominant negative and constitutively active forms of Rac1, RhoA and Cdc42 in Bonhoeffer stripe assays. Nasal neurite outgrowth on PTPmu was blocked by Cdc42-DN. Temporal repulsion to a PTPmu substrate was substantially reduced by addition of Cdc42-DN. The molecule that regulates the switch between permissive versus repulsive responses to PTPmu is Rac1 for temporal neurons. Inhibition of Rac1 is required for repulsion of temporal neurons. Interestingly, adding Rac1-CA to temporal RGC neurons converted PTPmu-dependent repulsion to a permissive response. In addition, adding exogenous Rac1-DN to nasal neurons induced a phenotype switch from a permissive to repulsive response to PTPmu. Together these data suggest that Cdc42 activity is required for both permissive and repulsive responses to PTPmu. However, the key to PTPmu-dependent repulsion is inhibition of Rac1 activity in temporal RGC neurons.

Protein-tyrosine phosphatase (PTP) wedge domain peptides: a novel approach for inhibition of PTP function and augmentation of protein-tyrosine kinase function.

Inhibition of protein-tyrosine phosphatases (PTPs) counterbalancing protein-tyrosine kinases (PTKs) offers a strategy for augmenting PTK actions. Conservation of PTP catalytic sites limits development of specific PTP inhibitors. A number of receptor PTPs, including the leukocyte common antigen-related (LAR) receptor and PTPmu, contain a wedge-shaped helix-loop-helix located near the first catalytic domain. Helix-loop-helix domains in other proteins demonstrate homophilic binding and inhibit function; therefore, we tested the hypothesis that LAR wedge domain peptides would exhibit homophilic binding, bind to LAR, and inhibit LAR function. Fluorescent beads coated with LAR or PTPmu wedge peptides demonstrated PTP-specific homophilic binding, and LAR wedge peptide-coated beads precipitated LAR protein. Administration of LAR wedge Tat peptide to PC12 cells resulted in increased proliferation, decreased cell death, increased neurite outgrowth, and augmented Trk PTK-mediated responses to nerve growth factor (NGF), a phenotype matching that found in PC12 cells with reduced LAR levels. PTPmu wedge Tat peptide had no effect on PC12 cells but blocked the PTPmu-dependent phenotype of neurite outgrowth of retinal ganglion neurons on a PTPmu substrate, whereas LAR wedge peptide had no effect. The survival- and neurite-promoting effect of the LAR wedge peptide was blocked by the Trk inhibitor K252a, and reciprocal co-immunoprecipitation demonstrated LAR/TrkA association. The addition of LAR wedge peptide inhibited LAR co-immunoprecipitation with TrkA, augmented NGF-induced activation of TrkA, ERK, and AKT, and in the absence of exogenous NGF, induced activation of TrkA, ERK, and AKT. PTP wedge domain peptides provide a unique PTP inhibition strategy and offer a novel approach for augmenting PTK function.

The receptor protein-tyrosine phosphatase PTPmu interacts with IQGAP1.

The receptor protein-tyrosine phosphatase PTPmu is a member of the Ig superfamily of cell adhesion molecules. The extracellular domain of PTPmu contains motifs commonly found in cell adhesion molecules. The intracellular domain of PTPmu contains two conserved catalytic domains, only the membrane-proximal domain has catalytic activity. The unique features of PTPmu make it an attractive molecule to transduce signals upon cell-cell contact. PTPmu has been shown to regulate cadherin-mediated cell adhesion, neurite outgrowth, and axon guidance. Protein kinase C is a component of the PTPmu signaling pathway utilized to regulate these events. To aid in the further characterization of PTPmu signaling pathways, we used a series of GST-PTPmu fusion proteins, including catalytically inactive and substrate trapping mutants, to identify PTPmu-interacting proteins. We identified IQGAP1, a known regulator of the Rho GTPases, Cdc42 and Rac1, as a novel PTPmu-interacting protein. We show that this interaction is due to direct binding. In addition, we demonstrate that amino acid residues 765-958 of PTPmu, which include the juxtamembrane domain and 35 residues of the first phosphatase domain, mediate the binding to IQGAP1. Furthermore, we demonstrate that constitutively active Cdc42, and to a lesser extent Rac1, enhances the interaction of PTPmu and IQGAP1. These data indicate PTPmu may regulate Rho-GTPase-dependent functions of IQGAP1 and suggest that IQGAP1 is a component of the PTPmu signaling pathway. In support of this, we show that a peptide that competes IQGAP1 binding to Rho GTPases blocks PTPmu-mediated neurite outgrowth.